This project will probe interactions at the active sites of a variety of members of the Aspartyl Protease class of enzymes. These studies will contribute to our understanding of the mechanism of action of these enzymes. They will reveal differences in active site geometry and in secondary specificity. These objectives will be accomplished by the synthesis and characterization of a series of new peptide of new peptide substrates and new peptide inhibitors for these enzymes. These compounds will be assembled by solid phase peptide synthesis, purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography, and characterized by peptide sequencing. The substrate series will have a -Phe-(NO2)Phe-reactive bond that has been shown to be hydrolyzed by these enzymes. The sequence of the octapeptide substrate and the inhbitors will be varied based on previous work with porcine pepsin and will include residues to enhance the solubilllty. The substrates will be examined by steady-state kinetics and by cryoenzymological techniques. The kinetic parameters of a variety of enzymes will be determined and the optimal substrate for each enzyme found. The inhibitors will be examined for this strength of binding to the various enzymes and for their kinetics of binding. Some of the inhibitors prepared will be designed to serve as transition state analogs for the Aspartyl Proteases. This design is based on a careful study of the active site features revealed by crystallographic methods.